Recombinant collagen hydrogels induced by disulfide bonds

Abstract With the characteristics of low toxicity and biodegradability, recombinant collagen‐like proteins have been chemically and genetically engineered as a scaffold for cell adhesion and proliferation. However, most of the existing hydrogels crosslinked with peptides or polymers are not pure collagen, limiting their utility as biomaterials. A major roadblock in the development of biomaterials is the need for high purity collagen that can self‐assemble into hydrogels under mild conditions. In this work, we designed a recombinant protein, S‐VCL‐S, by introducing cysteine residues into the Streptococcus pyogenes collagen‐like protein at both the N‐and C‐termini of the collagen with a trimerization domain (V) and a collagen domain (CL). The S‐VCL‐S protein was properly folded in complete triple helices and formed self‐supporting hydrogels without polymer modifications. In addition, the introduction of cysteines was found to play a key role in the properties of the hydrogels, including their microstructure, pore size, mechanical properties, and drug release capability. Moreover, two/three‐dimensional cell‐culture assays showed that the hydrogels are noncytotoxic and can promote long‐term cell viability. This study explored a crosslinking collagen hydrogel based on disulfide bonds and provides a design strategy for collagen‐based biomaterials.

has been studied in cartilage differentiation in vitro, opening the prospect of clinical applications for advanced treatment systems. 11 Moreover, collagen hydrogels have been explored in regenerating damaged corneal stroma in vivo and improving vascularization in islets. 15,16 However, natural collagen also has limitations, such as inconsistent purity and quality. It is an easy contamination source for various pathogens carried by animals. In addition, there is significant variability in hydrogel structure and mechanical properties in hydrogels prepared from animal-extracted type I collagen, depending on the tissue source and animal age, which impact cellular behavior.
To overcome these issues, recent advances in recombinant protein production have enabled the large-scale production of purified biological products. 18 Therefore, extensive research has focused on producing recombinant collagen-like proteins such as Streptococcal collagen-like protein 2 (Scl2), initially discovered in Streptococcus pyogenes (S. pyogenes), 19 which can be easily engineered upscaled, and purified. However, the utility of these recombinant collagen-like proteins is hindered by the difficulty of assembling them into stable 3D bioactive hydrogels.
These proteins are comprised of a trimerization domain (V) at the N-terminus and a collagen domain (CL), which forms a triple-helical structure with a melting point (Tm) around 37 C. [20][21][22] These collagenlike proteins have several advantages over traditional collagen hydrogels when used for hydrogels. These are high purity and good biocompatibility, biodegradability, molecular designability, and opening an avenue for safe biomedical materials without contamination from animal-derived diseases. 23 The utility of the recombinant collagen-like proteins is currently limited due to the inability of Scl proteins to assemble into stable 3D bioactive hydrogels. Bioactive hydrogels have been fabricated by combining functionalized Scl2 proteins with high molecular weight natural polymers or crosslinking agents. Such as poly (ethylene glycol) diacrylate, gelatin, fibrin, and polysaccharide-derived polymers, [24][25][26][27][28] and synthetic molecules, such as polymers of acrylic acid and ethylene oxide, and its derivatives. Moreover, short peptides, integrin-binding domains, heparin-binding domains, hyaluronic acid-binding sequences, chondroitin sulfate binding sequences, and matrix metalloproteinases cleavage siters have been employed for the preparation of responsive hydrogels. This was done by recombinant techniques for additional bioactivity to tune the cellular response finely. [29][30][31][32] However, the hydrogels formed by chemical polymerization appear to retain the properties of the polymer rather than the Scl2 protein itself, such as broad tunability and mechanical properties by varying the modified polymer concentration.
Self-assembling systems are highly desirable when compared with methods to modify polymers chemically. 33 Thus, specific chemical modifications, including cysteine and tyrosine residues, were introduced to bacterial collagen through covalent bonding and crosslinked with ruthenium [RuI(bpy) 3 ]Cl 2 , forming collagen gels. 34 However, metal crosslinking agents with weak biocompatibility and slow degradation rates need to be supplemented in vivo, 35,36 limiting their application in clinical research as biomaterials. Therefore, much research is still needed in the field of tissue engineering to endow pure collagen with the ability to self-assemble into hydrogels under mild conditions.
In this work, we utilized a genetic modification approach to generate collagen hydrogels by introducing cysteine residues at both the N-and C-termini of the S. pyogenes collagen-like gene and introducing a GFPGER motif in the collagen domain for cell adhesion. The pure recombinant collagen mutants self-assembled into hydrogels via disulfide bridge formation between the cysteines by redox reactions, eliminating the need for exogenous crosslinking agents or polymers. The structure of the collagen-like proteins and Tm values were characterized by circular dichroism. The network morphology of the hydrogels was examined by scanning electron microscopy (SEM) imaging, and the mechanical strength of the hydrogels was characterized by microrheology. Cell-culture and drug-release experiments demonstrated the potential application of hydrogels as biomaterials.

| Molecular design of the collagen polymers
Collagen sequences containing the GFPGER region of the bioactive structure were designed. Cysteine residues were introduced at the two end sites of the collagen sequence to enable oxidative crosslinking. The Gene Designer software was used to design the target protein. The design of protein polymers VCL and S-VCL-S are shown in Figure 1 A-B. Several considerations were made for optimal transcription and translation of the collagen encoding gene in Escherichia coli (E. coli) cells. These are (1) preferred codons in E. coli were selected. (2) High-frequency amino acids were encoded with multiple codons.
(3) Endonuclease cleaving sites were avoided in the designed sequence.
(5) The GC content of the nucleotide sequence was maintained at about 50%.

| Construction of plasmids
The S-VCL-S and VCL sequences were synthesized and cloned into the pET-28a vector by GENEWIZ, Inc. (Suzhou, China) for protein expression by double enzyme digestion with BamHI and NcoI (New England Biolabs, USA). For plasmid construction and amplification, E. coli DH5α cells and Luria-Bertani (LB) culture medium containing 1% tryptone, 0.5% yeast extract, and 1% sodium chloride were used.

| Recombinant protein expression and purification
The VCL and S-VCL-S proteins were overexpressed in E. coli BL21 (DE3) cells for protein expression. A single bacterial colony was selected for culture in 4 ml LB medium, supplemented with 50 μg/ml kanamycin, at 37 C overnight, shaking at 220 rpm. The overnight culture was diluted in fresh LB medium containing kanamycin to a ratio

| Formation of cysteine-based disulfide bond crosslinks
To form the disulfide bond crosslinks, 90 μl of 2%-4% (wt/vol) S-VCL-S solution was added to a glass tube. Then, 10 μl of hydrogen peroxide (H 2 O 2 ) (0.5%-1%) was added, mixed gently, and incubated in a water bath at 37 C for 30 min. In the control group, 10 μl of H 2 O 2 was added directly to 90 μl of 2%-4% (wt/vol) VCL and incubated at 37 C for 30 min and then left at 4 C for at least 7 days. After gel formation, the glass tube was removed and placed upside down. If the polymer molecules were crosslinked to form a hydrogel, their mobility was limited and remained at the bottom of the glass tube. However, gel formation could be detected when it flowed down the tube wall.
The observations were recorded with an ordinary photographic camera.

| Microrheology
Soft materials, such as gel solutions, which have complex structures with characteristic length and time scales, can be characterized by the stress relaxation modulus, G(ϖ). Herein, we added concentrated  A commercial polystyrene-coated cell culture plate was used as a control.

| Detection of cell proliferation
According to the manufacturer's instructions, cell viability was assessed using a CCK8 assay (Cell Counting Kit-8, Japan, ck04).   Figure 1A). To obtain hydrogels, H 2 O 2 was added to induce the formation of intermolecular disulfide bonds between the inserted cysteines ( Figure 1B). VCL was also tested as a negative control, characterized by Brodsky et al. 40 3.2 | Expression and purification of the designed collagen proteins

| Protein expression and purification
The collagen proteins, VCL and S-VCL-S, were overexpressed in E. coli  (Table S2).

| Secondary-structure characterization and thermal stability
To test whether the triple-helical domain of S-VCL-S has the proper structure, we characterized the secondary structure of the protein with CD spectroscopy on assemblies at a low concentration (1 mg/ml) for optical transparency. The CD spectrum from 240 to 200 nm of S-VCL-S showed typical triple-helical spectra with a positive peak at 220 nm and a minimum at 210 nm ( Figure 3A). The ellipticity difference between both proteins was small at 220 nm. The mean residue ellipticity value of S-VCL-S at 220 nm was closer to zero than VCL ( Figure 3A). These results confirmed that α-helices and triple helices were similarly formed for the S-VCL-S protein to VCL.
The thermal stability of S-VCL-S was determined by monitoring the intensity of the triple helix maximum (220 nm) at increasing temperatures at a rate of 0.1 C/min. The melting transitions of the S-VCL-S protein showed a sigmoidal shape, resembling collagen melting curves, and were consistent with VCL. The T m of S-VCL-S was around 37 C ( Figure 3B), consistent with the negative control (VCL) observed by Brodsky et al. 41 Notably, the protein with the cysteine residues was as stable as the native VCL, suggesting that adding cysteine residues to VCL may not disturb the formation of the secondary structures and the stability of the protein.

| Gelation
To investigate the response of the S-VCL-S protein to oxidative environments and its ability to form hydrogels, an inversion test was performed to characterize the hydrogels' properties using H 2  Notably, VCL could not be oxidized by H 2 O 2 at any concentration and remained as a solution even at concentrations up to 4% (wt/vol) ( Figure 4B). These results indicated that the disulfide bonds between cysteines were crucial for hydrogel formation.

| Microstructures of collagen hydrogels
To verify its effect on the microstructure of the recombinant collagen hydrogels, H 2 O 2 was added to S-VCL-S, and the porous microstructures were analyzed by SEM ( Figure 6). The S-VCL-S and VCL proteins were lyophilized before and after the addition of H 2 O 2 and imaged with SEM. After lyophilization, both S-VCL-S and VCL, without H 2 O 2 , formed a loose pore structure ( Figure 6A,D). After the addition of H 2 O 2 , S-VCL-S formed a relatively dense pore structure ( Figure 6B,C). However, no change was detected for VCL under the same conditions ( Figure 6D-F).
These results showed that the microstructure of S-VCL-S hydrogels with H 2 O 2 had a smaller pore size than those without H 2 O 2 .  F I G U R E 9 Sustained release of Rhodamine B from the hydrogels. A solution of 50 μg/ml Rhodamine B, 0.05% (wt/vol) H 2 O 2 , and 4% (wt/vol) S-VCL-S protein were mixed to form hydrogels at 37 C on a 96-well plate. Rhodamine B release was initiated by dropping PBS onto the hydrogel surface. PBS, phosphate-buffered saline (Rhodamine B) were used to determine the response of the hydrogels to the environment and observe the effect of hydrogels on the drug release rate. As shown in Figure 9, the cumulative release efficiency of Rhodamine B from S-VCL-S hydrogels was determined. The S-VCL-S hydrogels released 72.05% ± 3.32% of the Rhodamine B after 72 h in the release buffer, gradually released into the solution. These results demonstrated that the S-VCL-S collagen hydrogels could act as drug delivery platforms, which could meet the needs of biomaterials.

| DISCUSSION
Recombinant collagen-like proteins are now regarded as an ideal source for novel biomaterial products. For example, Scl2 can be easily fermented and purified on a large scale, 20 with consistent and welldefined production processes essential for pharmaceutical purposes.
However, Scl2 cannot self-assemble into gels as blank template protein, and the use of polymers and metal ions is currently not available in clinical practice. This study explored a method of crosslinking collagen hydrogels based on disulfide bonds. Its network morphology was examined by SEM and characterized with microrheology. The results demonstrated that hydrogels may have potential application as biomaterials and suggested that disulfide bonds can promote the crosslinking of collagen molecules to form hydrogels.
Self-assembling systems are highly desirable compared with methods to modify polymers chemically. 33  Previous studies have focused on specific chemical modifications, e.g., cysteine and tyrosine residues, for covalent bonding and crosslinking with ruthenium [RuI(bpy) 3 ]Cl 2 , that also successfully form collagen gels. 34 However, metal crosslinking agents with weak biocompatibility and slow degradation rates need to be supplemented in vivo, 35,36 limiting their applicability in clinical research as biomaterials. In contrast, the novel S-VCL-S construct here presented can be from a hydrogel structure under oxidative environments.
In the presence of 0.1% H 2 O 2 , a 4% (wt/vol) solution S-VCL-S self-assembles into a visible hydrogel, which can be checked macroscopically. This was further confirmed by microrheology but with a much lower concentration so samples could be easily injected into the capillary space. Hence, the values reported for the elastic moduli in the microrheology experiments were smaller than those expected from the tube-inverting experiments. Nonetheless, the formation of a hydrogel matrix was further validated and characterized by electron microscopy. In addition, the concentrations of H 2 O 2 and the S-VCL-S protein were found to play a key role in the hydrogel properties. A translucent gel could be formed under 4% (wt/vol) S-VCL-S and 0.1% (wt/vol) H 2 O 2 . However, if the protein concentration is less than 4% and H 2 O 2 is less than 0.05%, hydrogels could not be formed.
Biomaterials are frequently used as cell culture matrix 6 or drug delivery carrier. 7,8 S-VCL-S hydrogels could sustain MC3T3-E1 adhesion and growth. Cell growth seemed slightly impaired compared to controls, but this was true for both Scl2 protein and S-VCL-S. Ramshaw  S-VCL-S hydrogels could also act as drug carriers. Oxidativecrosslink S-VCL-S hydrogels could gradually release Rhodamine B for 72 h into the solution. Such release rates appear slower than some compounds, such as S4E8C hydrogels. However, it was higher than SE8C and S2E8C hydrogels, which might be related to their microstructures. 42 The ratio of Cys to the total number of residues is positively correlated with gel strength. Gel strength increases with the higher ratios, from 0.3% to 26.7%, whereas it decreases with lower ratios. 34,42,44,45 These results suggested that disulfide bonds can promote the crosslinking of collagen molecules to form hydrogels. In addition, the mechanical strength of the hydrogel increases with an increase in the crosslinking degree of disulfide bonds. Furthermore, gel stiffness increased according to the number of Cys residues; however, the stiffness reached a limit when the ratio of Cys to the total residue number was 20% or higher. 45 This study provides a means of chemical crosslinking collagen hydrogels based on disulfide bonds, which can be further optimized by adjusting the amount of cysteine to control the properties of the materials. This work could find broader applications in drug delivery and tissue engineering in the future.

| CONCLUSION
In this study, we developed a recombinant collagen-like protein, S-VCL-S, where Cys residues were inducted into the N and C-termini of the VCL domain. The S-VCL-S reacted with H 2 O 2 , a mild redox agent, to generate covalently crosslinked hydrogel through disulfide bridges between the cysteines by the oxidation reaction. The 2D/3D cellculture results showed that the hydrogels are noncytotoxic and can promote long-term cell viability. The hydrogels have typical mesh-like structure characteristics, significantly improve drug release capability, and have broad application prospects. It can sustain cell growth on a 2D and 3D cell culture model, sufficient for long-term cell viability.